Orai1 subunit stoichiometry of the mammalian CRAC channel pore - PubMed (original) (raw)

Orai1 subunit stoichiometry of the mammalian CRAC channel pore

Olivier Mignen et al. J Physiol. 2008.

Abstract

Agonist-activated Ca2+ entry plays a critical role in Ca2+ signalling in non-excitable cells. One mode of such entry is activated as a consequence of the depletion of intracellular Ca2+ stores. This depletion is sensed by the protein STIM1 in the endoplasmic reticulum, which then translocates to regions close to the plasma membrane where it induces the activation of store-operated conductances. The most thoroughly studied of these conductances are the Ca2+ release-activated Ca2+ (CRAC) channels, and recent studies have identified the protein Orai1 as comprising the essential pore-forming subunit of these channels. Although evidence suggests that Orai1 can assemble as homomultimers, whether this assembly is necessary for the formation of functional CRAC channels and, if so, their relevant stoichiometry is unknown. To examine this, we have used an approach involving the expression of preassembled tandem Orai1 multimers comprising different numbers of subunits into cells stably overexpressing STIM1, followed by the recording of maximally activated CRAC channel currents. In each case, any necessity for recruitment of additional Orai1 units to these preassembled multimers in order to form functional channels was evaluated by coexpression with a dominant-negative Orai1 mutant. In this way we were able to demonstrate, for the first time, that the functional CRAC channel pore is formed by a tetrameric assembly of Orai1 subunits.

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Figures

Figure 1

Figure 1. The E106Q mutant Orai1 inhibits CRAC channel currents in STIM1- stable cells stably expressing wild-type Orai1

A, inward CRAC channel currents measured at −40 mV in STIM1-stable cells stably expressing the wild-type Orai1 either alone (black bar) or after transfection with the E106Q mutant Orai1 (red bar). Data are presented as means ±

s.e.m.

; n = 15 and 10, respectively. B, representative current–voltage relationships for CRAC channel currents measured in STIM1-stable cells stably expressing wild-type Orai1 monomers either alone (black trace), or after transfection of the E106Q mutant Orai1 (red trace). C, upper panels, images showing the absence of significant background fluorescence in untransfected STIM1-stable cells. Shown is a DIC image of STIM1-stable cells (left panel), and the same field in confocal mode following immunocytochemistry with the anti-FLAG antibody (right panel). Lower panel, confocal image showing that transfection with the E106Q mutant Orai1 in STIM1-stable cells stably expressing the Orai1 monomer does not affect the plasma membrane localization of Orai1. Orai1 is detected in the confocal image by an antibody targeting the C-terminal FLAG tag and shown in green. Those cells transfected with the E106Q mutant are distinguished by cotransfection of a nuclear-targeted HcRed plasmid (pHcRed1-N1, Clontech, Palo Alto, CA, USA).

Figure 2

Figure 2. Predicted composition of expressed channel stoichiometries in cells coexpressing the E106Q mutant Orai1 and each of the tandem Orai1 multimers

Wild-type Orai1 subunits (blue circles) and E106Q Orai1 (black circles) are shown for each transfected multimer (dimer, trimer or tetramer), together with the predicted possible combinations of the transfected subunits based on the stoichiometry of functional CRAC channels being either a dimer, a trimer, or a tetramer. Those assemblies predicted to result in functional channels are shown with a central ‘pore’ in red, and predicted non-functional channels are represented with a pore in white. The consequent predicted overall effect on CRAC channel currents in the presence of the E106Q mutant is indicated. See text for details.

Figure 3

Figure 3. Effect of expression of tandem Orai1 multimers with and without the E106Q mutant Orai1

A, inward CRAC channel currents measured at −40 mV in untransfected STIM1-stable cells (white bar) and after transfection of the wild-type Orai1 multimers either alone (black bars) or along with 0.75 μg DNA of the E106Q mutant Orai1 (red bars). Data are presented as means ±

s.e.m.

; n = 9–13. B, representative current–voltage relationships for CRAC channel currents measured in STIM1-stable cells after transfection of the respective wild-type Orai1 multimers.

Figure 4

Figure 4. Expression of the E106Q mutant has no effect on CRAC channel currents in cells stably expressing the Orai1 tetramer

A, inward CRAC channel currents measured at −40 mV in STIM1- stable cells stably expressing the wild-type Orai1 tetramer either alone (black bar) or after transfection with the E106Q mutant Orai1 (red bar). Data are presented as means ±

s.e.m.

; n = 12 and 7, respectively. B, representative current–voltage relationships for CRAC channel currents measured in STIM1-stable cells stably expressing wild-type Orai1 tetramer either alone (black trace), or after transfection of the E106Q mutant Orai1 (red trace).

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